There is increasing evidence that cytokines, in general, and tumor necrosis factor (TNF), in particular, may play a key role in the pathogenesis of Crohn's disease (CD), one of the known idiopathic inflammatory bowel diseases (IBD)s. The most compelling evidence supporting the role of TNF in CD comes from recent studies using single dose infusions of anti- TNF monoclonal antibody in patients with active, therapy-refractory disease. In the majority of treated patients, TNF blockade resulted in decreased disease activity and sustained remission. Increasingevidence suggests that these effects are immunologically mediated. However, the precise mechanism(s) of TNF's actions in CD and the relationshipbetween TNF and immuneeffector cells remains unclear. Our preliminary findings provide direct evidence that a specific deregulation of TNF synthesis in mice, i.e. targeted deletion of AU-rich elements (AARE) in the in the 3'UTR region of the TNF gene, results in systemic TNF overexpression and triggers a CD-like intestinal phenotype that closely resembles the human disease. Therefore, the central hypothesis of this proposal is that TNF plays a crucial pathogenic role in CD by affecting primary immunoregulatory mechanisms, which result in the gut inflammatory changes characteristic of this disease. In this context, anti-TNF treatment may not directly target the effector functions of TNF but may, rather, interfere with these key immunologicalmechanisms. This hypothesis will be tested in TNFAARE mice by investigatingthe following 4 specific aims: 1) Characterize the features of chronic intestinal inflammation. The clinical phenotype, histological features, as well as the specific cytokine profiles will be investigated in our TNFAAR1;colony at UVA. 2) Define the role of the immune response in the development of intestinal inflammation. A series of cross-breeding studies, T-cell adoptive transfer as well as bone marrow chimera experiments will be performed to precisely define the role of T-cell subsets, B cells and other immunocytes in mediatingthe observed CD-like pathology. 3) Examine the role of the two TNF receptors in mediating intestinal inflammation. The expression of TNF receptor isoforms in intestinal tissues as well as isolated mucosal cells will be studied by multipletechniques. In addition, the AARE mutation will be introduced into TNFRI-or TNFRII-deficient mice to asssss the relative contributionof the two TNF receptors to TNF-mediated gut inflammation. Finally, we will study the effects of gut-specific reactivation of the TNFRI and/or TNFRII in double mutant TNFAAIUVTNFR 'mice. 4) Determine whether gut-specific TNF overexpression is sufficient to induce chronic intestinal inflammation. Using an intestinal-specificpromoter and Cre-loxP technology, we will compare the effects of systemic versus local TNF overexpression on the development of chronic intestinal inflammation in TNFAARH mice. The ultimate goal of the present research proposal is to define the precise mechanism(s) of TNF-induced CD in order to develop specific treatment modalities aimed at modifying the natural course of this devastating disease.